The ATM and Rad3-related (ATR) protein is a member of the phosphoinositide 3-kinase-related kinase (PIK) family. Members of this family play important roles in cell cycle regulation and DNA damage responses. Loss of functions mutations of the related ataxia telangiectasia mutated (ATM) protein lead to the devastating chromosomal instability disorder, AT. Studies of this disease have provided important insights into the cellular processes regulated by ATM. However, the functions of ATR in the maintenance of genomic stability in somatic cells are poorly understood. We have recently shown that ATR phosphorylates the tumor suppressor protein p53 at Ser15 and Ser37, which uncovers the specific transactivating function of this protein. These data support a role for ATR in the DNA damage-induced regulation of sequence-specific p53 DNA binding activity. The goals of this proposal are to improve our understanding of the cellular response to genotoxic stress by delineating the relevance of ATR activation to p53 function, and by defining the mechanisms whereby ATR is regulated in response to DNA damage. These objectives will be pursued by addressing the following specific aims: (1) to define regulatory interactions between ATR and the p53 pathway; and (2) to determine the activation requirements for the ATR kinase. These studies will provide new information regarding the mechanisms by which normal cells respond to genotoxic stress, and may yield new insights into the processes underlying the progression of human cancer.